Lyme disease is a bacterial infection caused by the spirochete Borrelia burgdorferi (B. burgdorferi) which is transmitted to patients through the bite of a blacklegged tick that remains attached to the patient for 24-36 hours. Symptomatically, early Lyme disease presents itself with flu-like symptoms causing the patient to experience body-wide itching, chills, fever, headache, muscle pain, and stiffness in the neck. Lyme disease is distinguished through the appearance of an erythema migrans, circular red rash around the bite area that resembles a “bull's eye”. Through early detection and immediate antibiotic treatment, Lyme disease can be completely cured. Although immediate antibiotic treatment has shown to completely cure patients of Lyme disease, the administration of the antibiotic treatment during the critical early stages of the disease is greatly dependent on an accurate diagnosis.
Misdiagnosis of Lyme disease often occurs as a result of a misinterpretation of the patient's clinical symptoms. In addition, Lyme disease exacerbates the diagnostic process by causing variability in the patient's immune response during the early stages of infection. The variability in the immune response can produce false negatives in serum antibody detection assays, such as immunofluorescent assays (IFA), enzyme-linked immunosorbent assays (ELISA), and Western Blot assays. These immunoassays utilize serum derived B. burgdorferi antigens to detect the presence of specific antibodies in a patient's body fluids. Due to the variability in the immune response during the early stages of infection, these antibody detection based assays are unable to detect the B. burgdorferi antibodies that are below a threshold concentration for detection. This inability to detect B. burgdorferi antibodies at the early stages of infection results in misdiagnosis that results in delayed administration of antibiotic treatments.
This antibody diagnostic detection approach has been found not to work as well with Lyme disease as with many other infectious diseases. One of the reasons for this is that only a low number of spirochetes is present and that it is hard for the immune system of the infected organism to detect the anitgens in the outer membrane of the spirochetes. Another reason is that the antibody response to the B. burgdorferi infection first arises weeks after the bite of the tick, and in many cases first after the patient has shown clinical signs of the disease.
An enzyme-linked immunosorbent assay (ELISA) has been used to detect antibodies in the blood of individuals with Lyme disease. This method uses a B. burgdorferi commercial antigen attached to a support. The antibody in the blood is reacted with this antigen and allowed to bind to the antigen. The resulting antigen-antibody is incubated with an enzyme-labeled anti-antibody which will bind to the antibody of the antigen-antibody complex. An enzyme substrate is then added. The enzyme on the anti-antibody will convert the substance to a product, and the amount of product is measured or detected, for example, visually. The enzyme activity of converting the substrate to a product is related to the amount of antibody bound to the antigen, thus indicating the amount of antibody in the blood and the presence of Lyme disease. Enzyme immunoassays have been desirable because the direct visualization of an antigen-antibody complex is thereby possible, using the product as a chromogenic indicator. This test can give false negatives if the concentration of antibodies is too low or the antibodies do not react well with the commercial antigens
A dot-immunobinding ELISA assay has also been developed. The principle of this type of assay is as follows: A dilute solution or suspension of commercial antigen is “dotted” on to a white, nitrocellulose piece of filter paper. The dot is then incubated with a first antibody from the blood of an infected patient and with a peroxidase enzyme-conjugated anti-antibody directed against the first antibody. After the enzymatic action of the peroxidase on a substrate, a product is formed and detected as a colored dot against the white filter paper background. This test can also give false negatives if the concentration of antibodies is too low or the antibodies do not react well with the commercial antigens.
Although it may take time for antibodies to develop in response to a B. burgdorferi infection, the presence of antigens in bodily fluids, such as blood and urine, may occur much sooner. These antigens are exported (or shed) in vivo and the detection of these antigens is a means of diagnosing Lyme disease. The antigens are extracellular membrane vesicles and other bioproducts including the major extracellular protein. Two well-known antigens that develop in response to a B. burgdorferi infection are designated as OspA and OspB. OspA has a molecular weight of 31 kDa and OspB 34 kDa. These antigens, however, do not appear early after infection. A 39 kDa antigen does appear early after infection.
U.S. Pat. No. 5,217,872 discloses a method for detection of B. burgdorferi antigens in bodily fluids, such as urine, as a diagnostic test for the presence of Lyme disease. In this method polyclonal antibodies were raised in rabbits against membrane vesicles and against an 83 kDa major extracellular protein (MEP) of B. burgdorferi. These antibodies were produced by rabbits in response to immunization with purified antigens, and the antibodies were purified by affinity chromatography. Immunized rabbits were periodically boosted with antigen suspended in dPBS. Sera were collected over a period of 10 weeks. Antibodies resulting from immunization with membrane vesicles served as capture antibodies. Antibodies resulting from immunization with MEP served as detection antibodies. The capture antibodies are bound to an inert solid support. A body fluid sample of an infected patient is brought into contact with this solid support under conditions conducive for the formation of immune complexes between the capture antibodies and antigens associated with B. burgdorferi in the body fluid sample. The solid support is washed and then brought into contact with detection antibodies under conditions conducive to formation of immune complexes consisting of the capture antibody, the antigens, and the detection antibody. The solid support with its immune complexes can be washed and then the antigen/capture antibody portion of the complex is detected by means well known in the art. Preferably, the detection antibody is conjugated with the enzyme horseradish peroxidase and detected by chromogenic assay. The capture antibodies recognize B. burgdorferi antigens at 11, 14, 22, 31, and 34 kDa in human urine in this assay. There is no evidence that the capture antibodies recognize the 39 kDa antigen in this assay.
It would be desirable to provide a diagnostic tool which is able to diagnose a B. burgdorferi infection at all stages, including at very early stages even before the clinical signs of infections appear. There exists a need for a reliable, rapid, inexpensive and non-invasive method for the diagnosis of Lyme disease. There are many situations in the diagnosis and treatment of Lyme disease where even a reliable test having a low level of false positives or negatives would be extremely valuable by itself, and particularly if used in conjunction with other tests that could be used to eliminate the false positives or negatives, or with clinical findings to identify the true positives.